The objective of the proposed research is to discover, characterize, and validate small molecule natural product-derived hypoxia-inducible factor-1 (HIF-1) inhibitors as adjunct agents for cancer therapy. The long-term goal is to develop cancer chemotherapeutic agents that specifically target tumor hypoxia (low oxygen tension). Solid tumors contain hypoxic regions and the extent of hypoxia correlates with malignant progression, resistance to radiation treatment and chemotherapy, and relapse of the disease. Hypoxia-associated treatment resistance can be caused directly by reduced cellular oxygen concentrations or indirectly by hypoxia-induced modifications in gene expression. We propose a new approach that specifically targets this important indirect effect of hypoxia (alteration of tumor gene expression). There is no approved drug in clinical use that specifically targets hypoxia. This drug discovery program uniquely combines natural products chemistry with cutting edge biomedical research. Specific Aim 1 will evaluate natural product-rich extracts, isolate and elucidate the chemical structures of active leads that inhibit hypoxia-induced and/or constitutively activated HIF-1, the principle transcription factor that activates the expression of adaptation and survival genes under hypoxia. Not only does HIF-1 promote hypoxic tumor survival and metastasis, oncogenic activation of HIF-1 is also associated with malignant progression and treatment resistance. Natural product-rich extracts will be examined in a panel of high-throughput bioassays for HIF-1 inhibitory activities (hypoxia-induced and oncogenically activated). Active compounds will be isolated through bioassay-guided isolation, and their structures elucidated using a combination of spectroscopic and spectrometric methods. Proof of principle has been established by the discovery of some of the most potent small molecule HIF-1 inhibitors. Specific Aim 2 will determine the effects of active compounds on hypoxic tumor cell survival, angiogenesis, and metastasis. A panel of tumor cell lines that represent different disease stages and hypoxia responsiveness will be used as in vitro models. Nontoxic active compounds that inhibit hypoxic tumor cell survival, angiogenesis, and metastasis in vitro will be further investigated in genetically engineered murine breast cancer models. Specific Aim 3 will investigate the therapeutic potential of natural product-derived HIF inhibitors as adjunct agents with chemotherapeutic agents. The goal is to identify the optimal combination of HIF-1 inhibitors with chemotherapy that can achieve the maximum efficacy of inhibiting tumor growth with low toxicity. Specific Aim 4 will characterize natural product-derived HIF inhibitors at the molecular and cellular levels and resolve the mechanisms of action. Accomplishing these objectives will provide antitumor drug leads and molecular probes that will afford new insights into the intracellular pathways that mediate hypoxic signaling.